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Traumatic injuries are among the leading causes of death and disability worldwide. Major trauma presentations have seen a demographic shift recently from the young to the elderly, with significant associated neurological deficit.To review the presentation and outcome of elderly patients presenting with cervical spinal injuries and associated neurological deficit that underwent surgical intervention in order to optimise treatment strategies.A retrospective review was conducted at a national tertiary referral centre to analyse admission trends from June 2016 to July 2020 for outcomes of elderly patients (≥ 65) presenting with traumatic cervical spine injuries associated with spinal cord injuries (SCI). Demographic, clinical, and radiological characteristics were collected and analysed.Forty-two patients met the inclusion criteria. The most common mechanisms of injury (MOIs) were falls from standing (38.1%) and falls from height (≥ 2 m) (33.3%). Complete SCIs had increased mean LOS (57.6 vs 21.6 days; p = 0.013), postoperative complications (100% vs 60.6%; p = 0.022), life-threatening complications (57.1% vs 9.1%; p = 0.001), and 90-day mortality (37.5% vs 5.9%; p = 0.007) compared to incomplete SCIs.Elderly patients with complete SCIs have poorer outcomes and mortality than those with less extensive SCIs. They require more resources, have greater risk of complications, and have higher mortality than those with incomplete SCIs, with subsequent implications on optimal treatment strategies. More robust studies are needed to derive improved risk stratification tools for geriatric patients with spinal injuries.

Background The SARS-CoV-2 pandemic has had profound implications on healthcare institutions. Aims This study aims to assess and compare referral patterns during COVID-19 to corresponding dates for the preceding 3 years (2017–2019), in order to preemptively coordinate the logistics of the surgical unit for similar future experiences. Methods Retrospective review for our institution, a national tertiary referral centre for spine pathology. Two distinct time-points were chosen to represent the varied levels of social restriction during the current pandemic: (i) study period 1 (SP1) from 11 November 2020 to 08 June 2020 represents a national lockdown, and (ii) study period 2 (SP2) from 09 June 2020 to 09 September 2020 indicates an easing of restrictions. Both periods were compared to corresponding dates (CP1: 11 March–08 June and CP2 09 June–09 September) for the preceding 3 years (2017–2019). Data collected included age, gender, and mechanism of injury (MOI) for descriptive analyses. MOIs were categorised into disc disease, cyclist, road-traffic-accident (RTA), falls 2 m, malignancy, sporting injuries, and miscellaneous. Results All MOI categories witnessed a reduction in referral numbers during SP1: disc disease (−29%), cyclist (−5%), RTAs (−66%), falls 2 m (−17%), malignancy (−33%), sporting injuries (−100%), and miscellaneous (−58%). Four of 8 categories (RTAs, falls 2 m) showed a further reduction (−34%, −27%) during SP2. One category (sporting injuries) portrayed a complete return to normal values during SP2 while a notable increase in cyclist-related referrals was witnessed (+ 63%) when compared with corresponding dates of previous years. Conclusion Spinal injury continues to occur across almost all categories, albeit at considerably reduced numbers. RTAs and falls remained the most common MOI. Awareness needs to be drawn to the reduction of malignancy-related referrals to dissuade people with such symptoms from avoiding presentation to hospital over periods of social restrictions.

Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage that is resistant to vascularization and endochondral ossification. During skeletal development articular cartilage also functions as a surface growth plate, which postnatally is replaced by a more spatially complex bone-cartilage interface. Motivated by this developmental process, the hypothesis of this study is that bi-phasic, fibre-reinforced cartilaginous templates can regenerate both the articular cartilage and subchondral bone within osteochondral defects created in caprine joints. To engineer mechanically competent implants, we first compared a range of 3D printed fibre networks (PCL, PLA and PLGA) for their capacity to mechanically reinforce alginate hydrogels whilst simultaneously supporting mesenchymal stem cell (MSC) chondrogenesis in vitro. These mechanically reinforced, MSC-laden alginate hydrogels were then used to engineer the endochondral bone forming phase of bi-phasic osteochondral constructs, with the overlying chondral phase consisting of cartilage tissue engineered using a co-culture of infrapatellar fat pad derived stem/stromal cells (FPSCs) and chondrocytes. Following chondrogenic priming and subcutaneous implantation in nude mice, these bi-phasic cartilaginous constructs were found to support the development of vascularised endochondral bone overlaid by phenotypically stable cartilage. These fibre-reinforced, bi-phasic cartilaginous templates were then evaluated in clinically relevant, large animal (caprine) model of osteochondral defect repair. Although the quality of repair was variable from animal-to-animal, in general more hyaline-like cartilage repair was observed after 6 months in animals treated with bi-phasic constructs compared to animals treated with commercial control scaffolds. This variability in the quality of repair points to the need for further improvements in the design of 3D bioprinted implants for joint regeneration. STATEMENT OF SIGNIFICANCE: Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage. In this study, we hypothesised that bi-phasic, fibre-reinforced cartilaginous templates could be leveraged to regenerate both the articular cartilage and subchondral bone within osteochondral defects. To this end we used 3D printed fibre networks to mechanically reinforce engineered transient cartilage, which also contained an overlying layer of phenotypically stable cartilage engineered using a co-culture of chondrocytes and stem cells. When chondrogenically primed and implanted into caprine osteochondral defects, these fibre-reinforced bi-phasic cartilaginous grafts were shown to spatially direct tissue development during joint repair. Such developmentally inspired tissue engineering strategies, enabled by advances in biofabrication and 3D printing, could form the basis of new classes of regenerative implants in orthopaedic medicine.

Tissue-specific extracellular matrix (ECM) proteins can play a key role in regulating the fate of stem cells and can potentially be utilized for therapeutic applications. Realising this potential requires further characterization of the diversity of biomolecules present in tissue-specific ECMs and an evaluation of their role as regulatory cues for regenerative medicine applications. The goal of this study was to identify specific soluble factors within the ECM of articular cartilage (AC) and growth plate (GP) that may impart chondro-inductivity or osteo-inductivity respectively. To this end, the significantly different proteins between both matrisomes were searched against the STRING database platform, from which C-type lectin domain family-11 member-A (CLEC11A) and S100 calcium-binding protein-A10 (S100A10) were identified as potential candidates for supporting osteogenesis, and Gremlin-1 (GREM1) and TGF-β induced gene human clone-3 (βIGH3) were identified as potential candidates for supporting stable chondrogenesis. Stimulation of chondrogenically-primed bone marrow-derived stem cells (BMSCs) with the AC-specific proteins GREM1 and βIGH3 had no noticeable effect on the deposition of collagen-II, a marker of chondrogenesis, but appeared to suppress the production of the hypertrophic marker collagen-X, particularly for higher concentrations of GREM1. Stimulation with GREM1 was also found to suppress the direct osteoblastic differentiation of BMSCs. In contrast, stimulation with the GP-specific factors CLEC11A and S100A10 was found to enhance osteogenesis of BMSCs, increasing the levels of mineralization, particularly for higher concentration of CLEC11A. Together these results demonstrate that AC- and GP-specific proteins may play a key role in developing novel strategies for engineering phenotypically stable articular cartilage or enhancing the regeneration of critically-sized bone defects.

In recent years, machine learning (ML) and artificial neural networks (ANNs), a particular subset of ML, have been adopted by various areas of healthcare. A number of diagnostic and prognostic algorithms have been designed and implemented across a range of orthopaedic sub-specialties to date, with many positive results. However, the methodology of many of these studies is flawed, and few compare the use of ML with the current approach in clinical practice. Spinal surgery has advanced rapidly over the past three decades, particularly in the areas of implant technology, advanced surgical techniques, biologics, and enhanced recovery protocols. It is therefore regarded an innovative field. Inevitably, spinal surgeons will wish to incorporate ML into their practice should models prove effective in diagnostic or prognostic terms. The purpose of this article is to review published studies that describe the application of neural networks to spinal surgery and which actively compare ANN models to contemporary clinical standards allowing evaluation of their efficacy, accuracy, and relatability. It also explores some of the limitations of the technology, which act to constrain the widespread adoption of neural networks for diagnostic and prognostic use in spinal care. Finally, it describes the necessary considerations should institutions wish to incorporate ANNs into their practices. In doing so, the aim of this review is to provide a practical approach for spinal surgeons to understand the relevant aspects of neural networks. Cite this article: Bone Joint J 2021;103-B(9):1442–1448.

Menisci play a major role in the mechanical function of the knee. They are subjected to large compressive forces and as a result and due to its avascular structure, menisci are prone to irreparable damage. Meniscectomy was once a common procedure for damaged menisci, however alternative approaches involving meniscus regeneration to restore function are of current interest. In order to enable these regenerative strategies, it is of utmost importance to initially establish he structure/property/function relationships of native menisci. Therefore, this study explores the influence of major constituents of the meniscal extracellular matrix; namely the glycosaminoglycan (GAG), and the collagen fibre orientation on the mechanical properties of the bovine meniscus. GAG distribution and mechanical properties are mapped with respect to depth and regional variance within the meniscus. Results show that the inner zone of the meniscus has a significantly larger quantity of GAG compared to the peripheral zone. The tibial and femoral layers contain a higher quantity of GAG than the mid-section and collagen fibre alignment differed depending on region. Overall, it was established that the viscoelastic properties of the meniscus are determined by the co-dependent relationship between the solid and fluid fractions of the meniscus and this varied depending on region. The hydrophilic nature of the GAG molecules play an important role in maintaining the solid/fluid balance while collagen fibre orientation restricts fluid flow within tissue, combined these processes act to support the meniscus under compressive loads.

Successfully treating osteochondral defects involves regenerating both the damaged articular cartilage and the underlying subchondral bone, in addition to the complex interface that separates these tissues. In this study, we demonstrate that a cartilage template, engineered using bone marrow-derived mesenchymal stem cells, can enhance the regeneration of such defects and promote the development of a more mechanically functional repair tissue. We also use a computational mechanobiological model to understand how joint-specific environmental factors, specifically oxygen levels and tissue strains, regulate the conversion of the engineered template into cartilage and bone in vivo.

Biological scaffolds generated from tissue-derived extracellular matrix (ECM) are commonly used clinically for soft tissue regeneration. Such biomaterials can enhance tissue-specific differentiation of adult stem cells, suggesting that structuring different ECMs into multi-layered scaffolds can form the basis of new strategies for regenerating damaged interfacial tissues such as the osteochondral unit. In this study, mass spectrometry is used to demonstrate that growth plate (GP) and articular cartilage (AC) ECMs contain a unique array of regulatory proteins that may be particularly suited to bone and cartilage repair respectively. Applying a novel iterative freeze-drying method, porous bi-phasic scaffolds composed of GP ECM overlaid by AC ECM are fabricated, which are capable of spatially directing stem cell differentiation in vitro, promoting the development of graded tissues transitioning from calcified cartilage to hyaline-like cartilage. Evaluating repair 12-months post-implantation into critically-sized caprine osteochondral defects reveals that these scaffolds promote regeneration in a manner distinct to commercial control-scaffolds. The GP layer supports endochondral bone formation, while the AC layer stimulates the formation of an overlying layer of hyaline cartilage with a collagen fiber architecture better recapitulating the native tissue. These findings support the use of a bi-layered, tissue-specific ECM derived scaffolds for regenerating spatially complex musculoskeletal tissues.

A range of bone regeneration strategies, from growth factor delivery and/or mesenchymal stem cell (MSC) transplantation to endochondral tissue engineering, have been developed in recent years. Despite their tremendous promise, the clinical translation and future use of many of these strategies is being hampered by concerns such as off target effects associated with growth factor delivery. Therefore the overall objective of this study was to investigate the influence of alpha-tricalcium phosphate (α-TCP) nanoparticle delivery into MSCs using an amphipathic cell penetrating peptide RALA, on osteogenesis in vitro and both intramembranous and endochondral bone formation in vivo. RALA complexed α-TCP nanoparticle delivery to MSCs resulted in an increased expression of bone morphogenetic protein-2 (BMP-2) and an upregulation in a number of key osteogenic genes. When α-TCP stimulated MSCs were encapsulated into alginate hydrogels, enhanced mineralization of the engineered construct was observed over a 28 day culture period. Furthermore, the in vivo bone forming potential of RALA complexed α-TCP nanoparticle delivery to MSCs was found to be comparable to growth factor delivery. Recognizing the potential and limitations associated with endochondral bone tissue engineering strategies, we then sought to explore how α-TCP nanoparticle delivery to MSCs influences early mineralization of engineered cartilage templates in vitro and their subsequent ossification in vivo. Despite accelerating mineralization of engineered cartilage templates in vitro, RALA complexed α-TCP nanoparticle delivery did not enhance endochondral bone formation in vivo. Therefore the potential of RALA complexed α-TCP nanoparticle delivery appears to be as an alternative to growth factor delivery as a single stage strategy for promoting bone generation.

Mimicking endochondral ossification to engineer constructs offers a novel solution to overcoming the problems associated with poor vascularisation in bone repair. This can be achieved by harnessing the angiogenic potency of hypertrophic cartilage. In this study, we demonstrate that tissue-engineered hypertrophically primed cartilage constructs can be developed from collagen-based scaffolds cultured with mesenchymal stem cells. These constructs were subsequently implanted into femoral defects in rats. It was evident that the constructs could support enhanced early stage healing at 4 weeks of these weight-bearing femoral bone defects compared to untreated defects. This study demonstrates the value of combining knowledge of development biology and tissue engineering in a developmental engineering inspired approach to tissue repair.

Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-γ-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bone marrow-derived mesenchymal stem cells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization and mineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.

Regenerating damaged tissue interfaces remains a significant clinical challenge, requiring recapitulation of the structure, composition and function of the native enthesis. In the ligament-to-bone interface this region transitions from ligament to fibrocartilage, to calcified cartilage and then to bone. This gradation in tissue types facilitates the transfer of load between soft and hard structures while minimizing stress concentrations at the interface. Previous attempts to engineer the ligament-bone interface have utilized various scaffold materials with an array of various cell types and/or biological cues. The primary goal of this study was to engineer a multiphased construct mimicking the ligament-bone interface by driving differentiation of a single population of mesenchymal stem cells (MSCs), seeded within blended fibrin-alginate hydrogels, down an endochondral, fibrocartilaginous or ligamentous pathway through spatial presentation of growth factors along the length of the construct within a custom-developed, dual-chamber culture system. MSCs within these engineered constructs demonstrated spatially distinct regions of differentiation, adopting either a cartilaginous or ligamentous phenotype depending on their local environment. Furthermore, there was also evidence of spatially defined progression towards an endochondral phenotype when chondrogenically primed MSCs within this construct were additionally exposed to hypertrophic cues. The study demonstrates the feasibility of engineering spatially complex soft tissues within a single MSC laden hydrogel through the defined presentation of biochemical cues. This novel approach represents a new strategy for engineering the ligament-bone interface. This article is protected by copyright. All rights reserved

Controlling the phenotype of mesenchymal stem cells (MSCs) through the delivery of regulatory genes is a promising strategy in tissue engineering (TE). Essential to effective gene delivery is the choice of gene carrier. Non-viral delivery vectors have been extensively used in TE, however their intrinsic effects on MSC differentiation remain poorly understood. The objective of this study was to investigate the influence of three different classes of non-viral gene delivery vectors: (1) cationic polymers (polyethylenimine, PEI), (2) inorganic nanoparticles (nanohydroxyapatite, nHA) and (3) amphipathic peptides (RALA peptide) on modulating stem cell fate after reporter and therapeutic gene delivery. Despite facilitating similar reporter gene transfection efficiencies, these nanoparticle-based vectors had dramatically different effects on MSC viability, cytoskeletal morphology and differentiation. After reporter gene delivery (pGFP or pLUC), the nHA and RALA vectors supported an elongated MSC morphology, actin stress fibre formation and the development of mature focal adhesions, while cells appeared rounded and less tense following PEI transfection. These changes in MSC morphology correlated with enhanced osteogenesis following nHA and RALA transfection and adipogenesis following PEI transfection. When therapeutic genes encoding for transforming growth factor beta 3 (TGF-β3) and/or bone morphogenic protein 2 (BMP2) were delivered to MSCs, nHA promoted osteogenesis in 2D culture and the development of an endochondral phenotype in 3D culture, while RALA was less osteogenic and appeared to promote a more stable hyaline cartilage-like phenotype. In contrast, PEI failed to induce robust osteogenesis or chondrogenesis of MSCs, despite effective therapeutic protein production. Taken together, these results demonstrate that the differentiation of MSCs through the application of non-viral gene delivery strategies depends not only on the gene delivered, but also on the gene carrier itself. Statement of Significance Nanoparticle-based non-viral gene delivery vectors have been extensively used in regenerative medicine, however their intrinsic effects on mesenchymal stem cell (MSC) differentiation remain poorly understood. This paper demonstrates that different classes of commonly used non-viral vectors are not inert and they have a strong effect on cell morphology, stress fiber formation and gene transcription in MSCs, which in turn modulates their capacity to differentiate towards osteogenic, adipogenic and chondrogenic lineages. These results also point to the need for careful and tissue-specific selection of nanoparticle-based delivery vectors to prevent undesired phenotypic changes and off-target effects when delivering therapeutic genes to damaged or diseased tissues.

Limitations associated with demineralised bone matrix and other grafting materials have motivated the development of alternative strategies to enhance the repair of large bone defects. The growth plate (GP) of developing limbs contain a plethora of growth factors and matrix cues which contribute to long bone growth, suggesting that biomaterials derived from its extracellular matrix (ECM) may be uniquely suited to promoting bone regeneration. The goal of this study was to generate porous scaffolds from decellularised GP ECM and to evaluate their ability to enhance host mediated bone regeneration following their implantation into critically-sized rat cranial defects. The scaffolds were first assessed by culturing with primary human macrophages, which demonstrated that decellularisation resulted in reduced IL-1β and IL-8 production. In vitro, GP derived scaffolds were found capable of supporting osteogenesis of mesenchymal stem cells via either an intramembranous or an endochondral pathway, demonstrating the intrinsic osteoinductivity of the biomaterial. Furthermore, upon implantation into cranial defects, GP derived scaffolds were observed to accelerate vessel in-growth, mineralisation and de novo bone formation. These results support the use of decellularised GP ECM as a scaffold for large bone defect regeneration.

The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-β3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.

Optic neuropathy is a disorder characterised by dysfunction or destruction of the optic nerve tissues. Acquired causes include interruption in the blood supply, nutritional deficiency, compression by a tumour or aneurysm, trauma, and toxic types (Ambizas and Patel In US Pharm 36(4):HS2–HS6, 1). Drug-induced optic neuropathy is of the toxic type and can be defined as a clinical syndrome characterised by papillomacular bundle damage, central, or cecocentral scotoma, and reduced colour vision (Ambizas and Patel In US Pharm 36(4):HS2–HS6, 2011; Sharma and Sharma In Indian J Ophthalmol 59(2):137–141, 2). To report a case unilateral optic neuritis, secondary to the use of the antiandrogen, cyproterone acetate. A 21-year-old female presented with a 4-day history of right brow pain exacerbated by eye movement, and blurring of the right temporal field of vision. She had been taking desogestrel 75 mg and cyproterone acetate 50 mg for the previous 2 months for hormone imbalance. Unaided right visual acuity measured 6/9 and unaided left visual acuity measured 6/6 on Snellen chart. Right red desaturation was present. Goldmann perimetry showed a right enlarged blind spot with predominantly temporal visual field loss. Visually evoked potential (VEP) testing of the right eye showed slightly increased latency, but normal amplitude. Three weeks after discontinuation of the antiandrogen therapy, her symptoms resolved. Repeat Goldmann visual fields showed expansion. Known side-effects of cyproterone acetate include retinal vascular disorder and retinal vein thrombosis, but an association with optic neuritis had not been described to date. There was a temporal relationship between cessation of the medication and improvement in visual symptoms. This implies that discontinuation of the offending drug constitutes the basis of treatment in drug-induced optic neuropathy.

The use of collagen-based scaffolds in orthopedic applications has been limited due to poor mechanical properties, but this may be overcome by the introduction of a stiffer supporting phase. Thus, we developed a synthesis technique to produce nonaggregating, stable nanohydroxyapatite (nHA) particles, permitting the fabrication of biomimetic-inspired scaffolds through the combination of nanosized HA with collagen, as found in native bone. This study evaluates the mechanical and biological impact of incorporating increasing concentrations of these nanoparticles into porous collagen scaffolds (1:1 and 5:1 weight ratios of nHA/collagen). Mechanical assessment demonstrated that increasing nHA incorporation correlated with increasing Young's moduli, which could be further amplified using cross-linking treatments. Typically, the porosity of a scaffold is sacrificed to produce a stiffer material; however, through the use of nanosized particles the inclusion of up to 5:1 nHA/collagen content still preserved the high 99% porosity of the composite scaffold, allowing for maximum cell infiltration. Moreover, increasing nHA presence induced significant bioactive responses, achieving superior cellular attachment and enhanced osteogenesis, promoting earlier expression of bone markers and cell-mediated mineralization versus nHA-free collagen controls. Interestingly, these content-dependent results observed in vitro did not directly translate in vivo. Instead, similar levels of bone formation were achieved within critical-sized rat calvarial defects, independent of nHA content, following acellular implantation. The addition of nHA, both 1:1 and 5:1, induced significantly higher levels of mineralization and de novo bone ingrowth versus collagen controls as demonstrated by microcomputed tomography, histological, and histomorphometric analyses. Ultimately, these results demonstrate the immense osteoinductivity of nonaggregated nanoparticles of HA incorporated into collagen-composite scaffolds and emphasize the importance of in vivo-based evaluation of therapies intended for clinical use.

Freshly isolated stromal cells can potentially be used as an alternative to in vitro expanded cells in regenerative medicine. Their use requires the development of bioactive hydrogels or scaffolds which provide an environment to enhance their proliferation and tissue-specific differentiation in vivo . The goal of the current study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM microparticles and transforming growth factor (TGF)-β3 as a putative therapeutic for articular cartilage regeneration. ECM microparticles were produced by cryomilling and freeze-drying porcine articular cartilage. Up to 2% (w/v) ECM could be incorporated into fibrin without detrimentally affecting its capacity to form stable hydrogels. To access the chondroinductivity of cartilage ECM, we compared chondrogenesis of infrapatellar fat pad-derived stem cells in fibrin hydrogels functionalized with either particulated ECM or control gelatin microspheres. Cartilage ECM particles could be used to control the delivery of TGF-β3 to IFP-derived stem cells within fibrin hydrogels in vitro , and furthermore, led to higher levels of sulphated glycosaminoglycan (sGAG) and collagen accumulation compared to control constructs loaded with gelatin microspheres. In vivo , freshly isolated stromal cells generated a more cartilage-like tissue within fibrin hydrogels functionalized with cartilage ECM particles compared to the control gelatin loaded constructs. These tissues stained strongly for type II collagen and contained higher levels of sGAGs. These results support the use of fibrin hydrogels functionalized with cartilage ECM components in single-stage, cell-based therapies for joint regeneration. Statement of Significance An alternative to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold or hydrogel is used to provide an environment that enhances their proliferation and tissue-specific differentiation in vivo . The objective of this study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM micro-particles and the growth factor TGF-β3 as a therapeutic for articular cartilage regeneration. This study demonstrates that freshly isolated stromal cells generate cartilage tissue in vivo when incorporated into such a fibrin hydrogels functionalized with cartilage ECM particles. These findings open up new possibilities for in-theatre, single-stage, cell-based therapies for joint regeneration.

Controlling the phenotype of transplanted stem cells is integral to ensuring their therapeutic efficacy. Hypoxia is a known regulator of stem cell fate, the effects of which can be mimicked using hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors such as dimethyloxalylglycine (DMOG). By releasing DMOG from mesenchymal stem cell (MSC) laden alginate hydrogels, it is possible to stabilize HIF-1α and enhance its nuclear localization. This correlated with enhanced chondrogenesis and a reduction in the expression of markers associated with chondrocyte hypertrophy, as well as increased SMAD 2/3 nuclear localization in the encapsulated MSCs. In vivo, DMOG delivery significantly reduced mineralisation of the proteoglycan-rich cartilaginous tissue generated by MSCs within alginate hydrogels loaded with TGF-β3 and BMP-2. Together these findings point to the potential of hypoxia mimicking hydrogels to control the fate of stem cells following their implantation into the body. STATEMENT OF SIGNIFICANCE: There are relatively few examples where in vivo delivery of adult stem cells has demonstrated a true therapeutic benefit. This may be attributed, at least in part, to a failure to control the fate of transplanted stem cells in vivo. In this paper we describe the development of hydrogels that mimic the effects of hypoxia on encapsulated stem cells. In vitro, these hydrogels enhance chondrogenesis of MSCs and suppress markers associated with chondrocyte hypertrophy. In an in vivo environment that otherwise supports progression along an endochondral pathway, we show that these hydrogels will instead direct mesenchymal stem cells (MSCs) to produce a more stable, cartilage-like tissue. In addition, we explore potential molecular mechanisms responsible for these phenotypic changes in MSCs.

Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.

Collagen is the main component of the extracellular matrix and has shown great promise as a biomaterial for tissue engineering and regenerative medicine applications. This chapter describes the structure and function of collagen and some of the manufacturing processes such as freeze-drying, electrospinning, and three-dimensional (3D) bioprinting that can be implemented to fabricate different 3D collagen-based biomaterials. Thereafter, various applications of these collagen-based biomaterials are discussed, focussing on their capabilities in promoting regeneration of different tissues, and their use as vehicles for the delivery of therapeutic agents including cells, growth factors, and genes. Finally, future trends in tissue engineering are investigated, and the role collagen-based biomaterials might play in guiding the field towards the ultimate goal of organ regeneration is explored.

Hypertrophic cartilaginous grafts can be engineered in vitro using bone marrow derived Mesenchymal Stem Cells (MSCs). When such engineered tissues are implanted in vivo they have been shown to induce bone formation by recapitulating aspects of the developmental process of endochondral ossification. Alginate, a naturally sourced and biocompatible hydrogel, offers an attractive 3D environment to facilitate the in vitro chondrogenesis of MSCs. Furthermore, such alginate hydrogels can potentially be used to engineer cartilage tissues of scale to promote endochondral bone regeneration in large bone defects. The aim of this study was to investigate the ability of chondrogenically-primed MSC-laden alginate hydrogels to induce healing in two distinct critically-sized defect models. Bone marrow derived MSCs were seeded into alginate hydrogels, chondrogenically primed in vitro in the presence of TGF-β3 and then implanted into either a critically-sized rat cranial or femoral defect. μCT analysis 4 weeks post-implantation revealed significantly higher levels of mineralization within the femoral defects treated with MSC-laden alginate hydrogels compared to untreated empty controls, with similar results observed within the cranial defects. However, any newly deposited bone was generated appositional to the alginate material, and occurred only superficially or where the alginate was seen to degrade. Alginate material was found to persist within both orthotopic locations 8 weeks post-implantation, with its slow rate of degradation appearing to prevent complete bone regeneration. In conclusion, while chondrogenically primed MSC–alginate constructs can act as templates to treat critically-sized defects within bones formed through either intramembranous or endochondral ossification, further optimization of the degradation kinetics of the hydrogel itself will be required to accelerate bone tissue deposition and facilitate complete regeneration of such defects.

An alternative strategy to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold is used to provide an environment conducive to proliferation and tissue-specific differentiation in vivo. The objective of this study is to develop a cartilage extracellular matrix (ECM)-derived scaffold that could facilitate the rapid proliferation and chondrogenic differentiation of freshly isolated stromal cells. By freeze-drying cryomilled cartilage ECM of differing concentrations, it is possible to produce scaffolds with a range of pore sizes. The migration, proliferation, and chondrogenic differentiation of infrapatellar fat pad-derived stem cells (FPSCs) depend on the concentration/porosity of these scaffolds, with greater sulphated glycosaminoglycan (sGAG) accumulation observed in scaffolds with larger-sized pores. It is then sought to determine if freshly isolated fat pad-derived stromal cells, seeded onto a transforming growth factor (TGF)-β3 eluting ECM-derived scaffold, could promote chondrogenesis in vivo. While a more cartilage-like tissue could be generated using culture expanded FPSCs compared to nonenriched freshly isolated cells, fresh CD44(+) stromal cells are capable of producing a tissue in vivo that stained strongly for sGAGs and type II collagen. These findings open up new possibilities for in-theatre cell-based therapies for joint regeneration.

The objective of this study was to develop a scaffold derived from cartilaginous extracellular matrix (ECM) that could be used as a growth factor delivery system to promote chondrogenesis of stem cells. Dehydrothermal crosslinked scaffolds were fabricated using a slurry of homogenized porcine articular cartilage, which was then seeded with human infrapatellar-fat-pad-derived stem cells (FPSCs). It was found that these ECM-derived scaffolds promoted superior chondrogenesis of FPSCs when the constructs were additionally stimulated with transforming growth factor (TGF)-β3. Cell-mediated contraction of the scaffold was observed, which could be limited by the additional use of 1-ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking without suppressing cartilage-specific matrix accumulation within the construct. To further validate the utility of the ECM-derived scaffold, we next compared its chondro-permissive properties to a biomimetic collagen-hyaluronic acid (HA) scaffold optimized for cartilage tissue engineering (TE) applications. The cartilage-ECM-derived scaffold supported at least comparable chondrogenesis to the collagen-HA scaffold, underwent less contraction and retained a greater proportion of synthesized sulfated glycosaminoglycans. Having developed a promising scaffold for TE, with superior chondrogenesis observed in the presence of exogenously supplied TGF-β3, the final phase of the study explored whether this scaffold could be used as a TGF-β3 delivery system to promote chondrogenesis of FPSCs. It was found that the majority of TGF-β3 that was loaded onto the scaffold was released in a controlled manner over the first 10days of culture, with comparable long-term chondrogenesis observed in these TGF-β3-loaded constructs compared to scaffolds where the TGF-β3 was continuously added to the media. The results of this study support the use of cartilage-ECM-derived scaffolds as a growth factor delivery system for use in articular cartilage regeneration.

The ability to print defined patterns of cells and extracellular-matrix components in three dimensions has enabled the engineering of simple biological tissues; however, bioprinting functional solid organs is beyond the capabilities of current biofabrication technologies. An alternative approach would be to bioprint the developmental precursor to an adult organ, using this engineered rudiment as a template for subsequent organogenesis in vivo. This study demonstrates that developmentally inspired hypertrophic cartilage templates can be engineered in vitro using stem cells within a supporting gamma-irradiated alginate bioink incorporating Arg-Gly-Asp adhesion peptides. Furthermore, these soft tissue templates can be reinforced with a network of printed polycaprolactone fibers, resulting in a ≈350 fold increase in construct compressive modulus providing the necessary stiffness to implant such immature cartilaginous rudiments into load bearing locations. As a proof-of-principal, multiple-tool biofabrication is used to engineer a mechanically reinforced cartilaginous template mimicking the geometry of a vertebral body, which in vivo supported the development of a vascularized bone organ containing trabecular-like endochondral bone with a supporting marrow structure. Such developmental engineering approaches could be applied to the biofabrication of other solid organs by bioprinting precursors that have the capacity to mature into their adult counterparts over time in vivo.

Collagen and collagen-based scaffolds offer distinct advantages when selected as biomaterials for use across a broad spectrum of regenerative medicine applications. However, relatively poor mechanical properties are often perceived to limit their usefulness for orthopedic applications. These problems can be overcome through enhanced crosslinking mechanisms or through the addition of a second, stiffer phase such as hydroxyapatite, thus allowing tailored composite scaffolds to meet specific tissue requirements. This overview will highlight the current state of the art of these scaffolds, and consider the exciting prospects and future directions of collagen-based technologies for orthopedic regenerative medicine.

The synthesis of nanophase hydroxyapatite (nHA) is of importance in the field of biomaterials and bone tissue engineering. The bioactive and osteoconductive properties of nHA are of much benefit to a wide range of biomedical applications such as producing bone tissue engineered constructs, coating medical implants, or as a carrier for plasmid DNA in gene delivery. This study aimed to develop a novel low-temperature dispersant-aided precipitation reaction to produce nHA particles (

Clinical translation of tissue engineered therapeutics is hampered by the significant logistical and regulatory challenges associated with such products, prompting increased interest in the use of decellularized extracellular matrix (ECM) to enhance endogenous regeneration. Most bones develop and heal by endochondral ossification, the replacement of a hypertrophic cartilaginous intermediary with bone. The hypothesis of this study is that a porous scaffold derived from decellularized tissue engineered hypertrophic cartilage will retain the necessary signals to instruct host cells to accelerate endogenous bone regeneration. Cartilage tissue (CT) and hypertrophic cartilage tissue (HT) were engineered using human bone marrow derived mesenchymal stem cells, decellularized and the remaining ECM was freeze-dried to generate porous scaffolds. When implanted subcutaneously in nude mice, only the decellularized HT-derived scaffolds were found to induce vascularization and de novo mineral accumulation. Furthermore, when implanted into critically-sized femoral defects, full bridging was observed in half of the defects treated with HT scaffolds, while no evidence of such bridging was found in empty controls. Host cells which had migrated throughout the scaffold were capable of producing new bone tissue, in contrast to fibrous tissue formation within empty controls. These results demonstrate the capacity of decellularized engineered tissues as ‘off-the-shelf’ implants to promote tissue regeneration.

The ability of nano-hydroxyapatite (nHA) particles developed in-house to act as non-viral delivery vectors is assessed. These nHA particles are combined with collagen to yield bioactive, biodegradable collagen nano-hydroxyapatite (coll-nHA) scaffolds. Their ability to act as gene-activated matrices for BMP2 delivery is demonstrated with successful transfection of mesenchymal stem cells (MSCs) resulting in high calcium production.

Bone regeneration requires scaffolds that possess suitable mechanical and biological properties. This study sought to develop a novel collagen-nHA biocomposite scaffold via two new methods. Firstly a stable nHA suspension was produced and added to a collagen slurry (suspension method), and secondly, porous collagen scaffolds were immersed in nHA suspension after freeze-drying (immersion method). Significantly stronger constructs were produced using both methods compared to collagen only scaffolds, with a high porosity maintained (98.9%). It was found that Coll-nHA composite scaffolds produced by the suspension method were up to 18 times stiffer than the collagen control (5.50 +/- 1.70 kPa vs. 0.30 +/- 0.09 kPa). The suspension method was also more reproducible, and the quantity of nHA incorporated could be varied with greater ease than with the immersion technique. In addition, Coll-nHA composites display excellent biological activity, demonstrating their potential as bone graft substitutes in orthopaedic regenerative medicine.